Differential regulation of bovine pyruvate carboxylase promoters by fatty acids and peroxisome proliferator-activated receptor-α agonist

Pyruvate carboxylase (PC) is a critical enzyme in supplying carbon for gluconeogenesis and oxaloacetate for the tricarboxylic acid cycle. The bovine PC (EC 6.4.1.1) gene contains 3 promoter sequences (P3, P2, and P1 from 5′ to 3′). Physiological stressors, including the onset of calving and feed restriction, lead to elevated nonesterified fatty acids and glucocorticoid levels that coincide with an increase in PC mRNA expression. The effects of elevated fatty acids on bovine PC mRNA expression and promoter function have not been determined. The objective of this experiment was to determine the direct effects of stearic, oleic, and linoleic acids, dexamethasone, and Wy14643 (a peroxisome proliferator-activated receptor-α agonist) on bovine PC promoter activity. Promoter-luciferase constructs, containing 1,005 bp of P1, 1,079 bp of P2, or 1,010 bp of P3, were transiently transfected into rat hepatoma (H4IIE) cells. Cells were then treated with 1 mM stearic, oleic, or linoleic acids, 1 μM dexamethasone, or 10 μM Wy14643 for 23 h. Activity of P1 was suppressed with exposure to stearic acid (1.58 vs. 6.19 ± 0.81 arbitrary units for stearic vs. control, respectively) and enhanced with exposure to Wy14643 (9.26 vs. 6.19 ± 0.81 arbitrary units for Wy14643 vs. control, respectively). Conversely, stearic acid enhanced P3 activity (2.55 vs. 0.40 ± 0.33 arbitrary units for stearic vs. control, respectively). Dexamethasone, linoleic acid, and oleic acid failed to elicit a response from any of the promoters tested. These data demonstrate the direct role of fatty acids in regulating PC expression and indicate that fatty acids provide promoter-specific regulation of PC promoters.     

Mammary gland expression of antibacterial peptide genes to inhibit bacterial pathogens causing mastitis

As a step toward prevention of bovine mastitis, a plasmid-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete bovine lactoferricin and bovine tracheal antibacterial peptides. For this purpose, a series of mammary tissue-specific expression vectors, harboring the antibacterial peptide gene, the 5′-flanking regulation sequence of goat β-casein, and the bovine growth hormone polyadenylation signal sequence, were constructed using a eukaryotic expression vector pIRES1-neo. The mammary gland tissue-specific expression vector carrying the antimicrobial peptide genes dissolved in physiologic saline was injected directly into the lactating mammary glands of goats. The milk samples after injection were checked by Tricine-SDS-PAGE and bacterium inhibition zone assay. The results of these tests showed that the mammary gland tissue-specific expression vector driven by the goat β-casein gene promoter could efficiently direct the expression of antibacterial peptides in goat milk; the expression of antibacterial proteins lasted for 3 to 6 d. All of the milk samples collected from the mammary glands that had been injected with different vectors harboring the antibacterial peptide gene(s) exhibited bacteriostatic activity against different bacterial pathogens. These results demonstrated that the mammary gland tissue-specific expression vector could be used to introduce antibacterial peptide gene into the goat mammary gland, enabling secretion of a bioactive form of antibacterial peptide in the milk. This successful expression of antibacterial peptides in goat mammary glands provided a possible method to prevent mastitis in ruminants.     

Expression of growth hormone receptor 1A messenger ribonucleic acid in liver of dairy cows during lactation and after administration of recombinant bovine somatotropin.

The mRNA for growth hormone receptor is transcribed from at least three different promoters in cattle. The first promoter (P1) is liver-specific and transcribes growth hormone receptor mRNA containing exon 1A (growth hormone receptor 1A). The second and third promoters (P2 and P3) are active in a variety of tissues and transcribe growth hormone receptor mRNA containing exon 1B and 1C. The objective was to characterize P1 activity by measuring the amount of growth hormone receptor 1A mRNA in liver of dairy cows at different stages of lactation as well as after administration of recombinant bovine somatotropin (rbST). In study 1, liver RNA was isolated from Holstein cows during the dry period (nonlactating, n = 6) and during early (n = 6), mid (n = 6), and late (n = 11) stages of lactation. Six of the late-lactation cows received injections of rbST (25 mg/d) for 7 d prior to collection of liver tissue. In study 2, lactating Holstein cows received either no infusion (control, n = 10) or continuous infusion of rbST (29 mg/d, n = 10) for 63 d. The amount of growth hormone receptor 1A mRNA was decreased in early- and mid-lactation cows compared with late-lactation cows or nonlactating cows (study 1). Administration of rbST increased growth hormone receptor 1A mRNA (studies 1 and 2). The total amount of growth hormone receptor transcribed from alternative promoters (growth hormone receptor P2 and P3) remained unchanged during different stages of lactation or in response to rbST. We conclude that changes in liver growth hormone receptor mRNA in lactating dairy cattle primarily depend on growth hormone receptor P1 activity.     

Expression of env sequences of the bovine leukemia virus (BLV) in the yeast Saccharomyces cerevisiae

DNA sequences of the envelope (env) gene of the bovine leukemia virus (BLV) were expressed in the yeast Saccharomyces cerevisiae. Two yeast promoters, the repressible PHO5 promoter and the constitutive PGK promoter, were used to construct four expression plasmids comprising either a sequence of the surface antigen gp51 or a (gp51 + gp30) sequence. The expressed heterologous gene products were characterized by Western blot analysis and competitive radioimmunoassay. By means of Northern blot analysis the steady-state level of env-specific mRNA was analysed. The highest expression rate was obtained from recombinant plasmid YEpSG 94 comprising a gp51 sequence--a 630 base pair fragment containing 70% of the gp51 but lacking the N terminus--as well as the PHO5 promoter including PHO5 signal sequence and the PHO5 terminator. The recombinant gp51 was partially glycosylated but the PHO5 signal peptide did not seem to be cleaved off. No immunoreactive material could be found in the periplasm or in the culture medium. By means of monoclonal antibodies directed against eight different epitopes of viral gp51, all four sequential antigenic determinants were detected in the AH 216 (YEpSG 94) expression product.     

Gluconeogenic enzymes are differentially regulated by fatty acid cocktails in Madin-Darby bovine kidney cells

Expression of mRNA for pyruvate carboxylase (PC) is elevated at calving and during other physiological states when plasma NEFA concentrations are increased. The objective of this study was to determine the direct effects of fatty acids on expression of PC, cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), mitochondrial PEPCK (PEPCK-M), and glucose-6-phosphatase (G6Pase) mRNA in Madin-Darby bovine kidney (MDBK) cells. Combinations of C14:0, C16:0, C18:0, C18:1n-6 cis, C18:2n-6 cis, and C18:3n-3 cis were created to mimic the profiles and concentrations in serum from far-off dry cows and late postcalving intervals (PRPT), the profile at calving (CALV), and the profile observed in cows induced to express fatty liver at calving (IFL). The MDBK cells were exposed to fatty acid mixtures for 24h at the following concentrations: 0.25 and 0.5mM for PRPT; 0.25, 0.5, and 1.0mM for CALV; and 0.5 and 1.0mM for IFL. Cells exposed to PRPT had greater PEPCK-C and tended to have greater G6Pase mRNA than control cells. Exposure of cells to 0.25mM PRPT increased expression of PEPCK-C compared with cells exposed to 0.5mM PRPT. Expression of PC and PEPCK-M did not differ with exposure to PRPT. Expression of PEPCK-C was decreased and that of PEPCK-M and G6Pase mRNA increased linearly in response to CALV. The ratio of PC:PEPCK-C mRNA was increased by the IFL mixture and in response to increasing amounts of the CALV fatty acid mixture. Treatment of cells with CALV or IFL increased the sum of PC 5' untranslated region (UTR) variants A, B, C, and F but did not alter PC 5' UTR D and E expression. The changes in PEPCK-C, G6Pase, and PC mRNA and the ratio of PC:PEPCK-C observed in MDBK cells in response to fatty acids suggests a role for fatty acid concentration and profile in mediating the expression of key gluconeogenic enzymes.     

Systematic transient assays of promoter activities for leaf-specific genes identified by gene-expression profiling with cDNA microarrays in Arabidopsis thaliana

We identified 85 genes highly expressed in leaves using an Arabidopsis cDNA microarray. A vector, pRAB5, was designed to allow cloning and assaying of the promoters. Fifty-one promoters from the selected genes were cloned and then assayed using a microprojectile bombardment and dual luciferase reporter assay system. This system allowed efficient systematic assays of promoter activity.     

Associations between novel single nucleotide polymorphisms in the Bos taurus growth hormone gene and performance traits in Holstein-Friesian dairy cattle

Growth hormone, produced in the anterior pituitary gland, stimulates the release of insulin-like growth factor-I from the liver and is of critical importance in the control of nutrient utilization and partitioning for lactogenesis, fertility, growth, and development in cattle. The aim of this study was to discover novel polymorphisms in the bovine growth hormone gene (GH1) and to quantify their association with performance using estimates of genetic merit on 848 Holstein-Friesian AI (artificial insemination) dairy sires. Associations with previously reported polymorphisms in the bovine GH1 gene were also undertaken. A total of 38 novel single nucleotide polymorphisms (SNP) were identified across a panel of 22 beef and dairy cattle by sequence analysis of the 5′ promoter, intronic, exonic, and 3′ regulatory regions, encompassing approximately 7 kb of the GH1 gene. Following multiple regression analysis on all SNP, associations were identified between 11 SNP (2 novel and 9 previously identified) and milk fat and protein yield, milk composition, somatic cell score, survival, body condition score, and body size. The G allele of a previously identified SNP in exon 5 at position 2141 of the GH1 sequence, resulting in a nonsynonymous substitution, was associated with decreased milk protein yield. The C allele of a novel SNP, GH32, was associated with inferior carcass conformation. In addition, the T allele of a previously characterized SNP, GH35, was associated with decreased survival. Both GH24 (novel) and GH35 were independently associated with somatic cell count, and 3 SNP, GH21, 2291, and GH35, were independently associated with body depth. Furthermore, 2 SNP, GH24 and GH63, were independently associated with carcass fat. Results of this study further demonstrate the multifaceted influences of GH1 on milk production, fertility, and growth-related traits in cattle.     

Hot topic: Gene duplication at the α-lactalbumin locus: Finding the evidence in water buffalo (Bubalus bubalus L.)

Studies on milk proteins revealed that a qualitative and quantitative polymorphism may often be found regarding α-lactalbumin (α-LA). In mammals, a similar phenomenon was widely documented in the α-globin system as the result of a gene duplication. The presence of several differently expressed α-lactalbumin gene (LALBA) products suggests that the mechanism underlying this phenomenon may involve nonallelic genes. To check this hypothesis, an experiment was set up to investigate the LALBA gene arrangement of a water buffalo exhibiting an α-LA phenotype characterized by a double-band pattern on PAGE isoelectric, focusing analysis of milk protein. In particular, the relative amount of protein inferred from the different intensity of the bands was consistent with a gene duplication. Thus, leukocyte DNA was extracted from a blood sample of the buffalo and amplified with 4 primers (2 RV-IVFW for PCR and 4 FW-IRV for nested PCR). The intergenic segments of the assumed duplicated gene were then amplified with 2 different PCR protocols. First, the segment limited by the third exon in the upstream gene and the second exon in the downstream gene was amplified by simple PCR, which gave aspecific results. Second, this PCR product was subjected to nested PCR, amplifying the segment limited by the fourth exon in the upstream gene and the first exon in the downstream gene, yielding an amplified nucleotide fragment of about 6,200 bp. Blood samples from an additional 15 buffalos were then analyzed in the same manner. The results obtained from the new samples confirmed the presence of an amplified nucleotide fragment of about 6,200 bp in most of them, though they all were characterized by an α-LA monomorphic phenotype. A couple of 6,200-bp fragments obtained were purified, cloned in pGEM-T easy vector system (Promega, Madison, WI) and sequenced. The sequence of the large DNA segments, containing the intergenic portion, was aligned with the LALBA gene (accession number AF194373; http://www.ncbi.nlm.nih.gov/Database/index.html). They both were found to coincide with the portion containing exon 4 and the untranslated region at the 3′ end of the upstream gene and with the portion containing exon 1 and the untranslated region at the 5′ end of the downstream gene. These results confirm the hypothesis that a tandemly repeated copy of the LALBA gene is present in water buffalo.     

Association of the OLR1 gene with milk composition in Holstein dairy cattle

Oxidized low-density lipoprotein receptor (OLR1) is the major protein that binds, internalizes, and degrades oxidized low-density lipoprotein. The role of OLR1 in lipid metabolism and the results of previous whole-genome scan studies prompted the investigation of OLR1 as a candidate gene affecting milk composition traits. Direct cDNA and genomic sequencing of OLR1 revealed 2 single nucleotide polymorphisms (SNP) in exon 4, 5 SNP in intron 4, and 1 in the 3′ untranslated region (UTR). Four intragenic haplotypes comprising SNP positions 7,160, 7,161, 7,278, 7,381, 7,409, 7,438, 7,512, and 8,232 were inferred. Haplotype analysis showed that one of the haplotypes was associated with a significant increase in fat yield and fat percentage. Single SNP analysis showed that allele C of SNP 8,232 (in the 3′-UTR) had significant effects on fat yield and fat percentage, whereas SNP 7,160 and 7,161 (in exon 4) had no significant effects. Both single SNP and haplotype analyses indicate that SNP 8,232 in the 3′-UTR is associated with milk fat yield and percentage and it may be in linkage disequilibrium with the functional polymorphism. To provide support for the hypothesis that SNP 8,232 is responsible for OLR1 expression, OLR1 expression levels in individuals bearing different genotypes were assessed. It was found that OLR1 expression was reduced in genotype AA individuals compared with CC and AC individuals, suggesting that A at position 8,232 may be the nucleotide causing decreased OLR1 expression. The 3′-UTR polymorphism found in this study might control translation or stability of OLR1 mRNA.     

Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the α_s1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5′ untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (α_s1-, α_s2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln₇₇→Pro_77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val₁₅ and AGA→AGG in Arg₁₀₀ of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5′ UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of α_s1-CN (15.32 g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln₇₇→Pro₇₇ was significantly higher than in milks of heterozygous (CA; 9.05 g/kg) and reference (AA; 7.61 g/kg) genotype animals. The α_s2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the sequence corresponded to allele A and presumably derived from it. Therefore, this allele is denoted by A₃. All goats from the reference genotype (AA) were homozygous for the allele at nucleotide position 1374 and 1866, whereas all mutations in the 5′ UTR existed in a heterozygous form in both heterozygous (CA) and the new mutation (CC) genotype. The newly identified mutation (CC) detected in some of the goat breeds is, therefore, important in selection for genetic improvement and high-quality milk for the emerging goat cheese-producing industries. The finding will also benefit farmers raising these goat breeds due to the increased selling price of goats. Further studies should investigate the effect of this amino acid exchange on the secondary and tertiary structure of the α_s1-CN molecule and on the susceptibility of peptide hydrolysis by digestive enzymes.     

重组普鲁兰酶在枯草芽孢杆菌中的高效表达

普鲁兰酶可特异性地水解支链淀粉得到直链淀粉,因而在淀粉加工过程中具有重要的应用。本研究从Bacillus naganoensis ATCC53909基因组中克隆了普鲁兰酶基因pul,并克隆到大肠杆菌-枯草芽孢杆菌穿梭载体p BE中,构建表达载体p BE-pul。在此基础上,将来源于枯草芽孢杆菌、地衣芽孢杆菌以及解淀粉芽孢杆菌中的17个高表达基因的启动子分别克隆到表达载体p BE-pul中,并转化至Bacillus subtilis ATCC6051?10,成功构建了十七株含有不同启动子介导普鲁兰酶分泌表达的重组菌株。对重组菌株的分泌表达比较发现,启动子P43和Pspov G介导的普鲁兰酶活性明显优于其他启动子,其中Pspov G介导的普鲁兰酶活性更高。同时,还使用了启动子Pspov G介导N端的108个氨基酸缺失的pul324突变体进行分泌表达。通过对17种启动子的比较和两个普鲁兰酶基因的比较,本研究构建的一株重组菌株的普鲁兰酶的表达更为高效,其活性高达389.85 U/mL,后者显著高于现有的相关报道。     

Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.